Method for quantification of biological material in a sample

ABSTRACT

Method for detection of a biological material in a sample. The method includes the steps of liquifying the sample (if necessary) and pouring the liquified sample into the incubation plate. The incubation plate has a generally flat horizontal surface and the surface is divided into a plurality of at least 20 recessed wells. Each well is adapted to hold an aliquot of liquid and is sized and shaped, and formed of a suitable material, to hold the aliquot within the well by surface tension. Any excess liquid from the liquified sample is poured from the surface of the plate. The method then involves incubating that incubation plate until the presence or absence of the biological material is determined.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Croteau et al.,U.S. Ser. No. 08/606,229 filed Feb. 23, 1996, which is acontinuation-in-part of Croteau et al., U.S. Ser. No. 08/557,529, filedNov. 13, 1995, both entitled “Method for Quantification of BiologicalMaterial in a Sample” hereby incorporated herein by reference, includingdrawings.

FIELD OF THE INVENTION

[0002] This invention relates to a method for quantification ofbiological material in a sample.

BACKGROUND OF THE INVENTION

[0003] Many industries need to detect and quantify the concentration andlevel of biological material in a sample. For example, the determinationof bacterial concentration in food and water is an essential part offood and water quality testing. EPA regulations require that no Coliformsuch as Escherichia coli can be present in potable water. The“presence/absence” format of a testing medium, such as Colilert®chemical mixture (IDEXX Laboratories, ME) which is used as a testingmedium for Escherichia coli and all coliform bacteria, is very useful inmaking this determination. Colilert® chemical mixture is based on theDefined Substrate Technology described in Edberg, “Method and Medium foruse in Detecting Target Microbes In Situ in A Specimen Sample of APossibly Contaminated Material,” U.S. Pat. Nos. 4,925,789 and 5,492,933.See also, Townsend et al., U.S. Ser. No. 08/484,593 filed Jun. 7, 1995entitled, “Method and Composition for Detecting Bacterial Contaminationin Food Products”, hereby incorporated by reference herein, describes amedium for the detection of bacteria in food and water samples.

[0004] However, there are areas where the quantification, not just thedetection, of bacterial concentration is important. Examples of suchareas include waste water, incoming water in water purification systems,surface water, and food testing. For example, numerous restaurant chainswill only accept raw ground beef or poultry that contains less than acertain concentration of bacterial contamination. Therefore, foodprocessing plants must carry out the necessary microbiological tests todetermine the. bacterial concentration of these food items before theycan be released to customers.

[0005] The classical methods of quantification of biological materialare the standard plate count method or the multiple tube fermentation(MTF) method. A quantity of sample being tested for microbialcontamination is first dispensed in a Petri dish. Then 15 ml of theappropriate media is poured over the sample. The Petri-dish is thenswirled to mix the sample in the medium and the Petri-dish is left tosolidify at room temperature for approximately 20 minutes. The medium isthen incubated at a specific temperature for a specific time, and anyresulting colonies are counted.

[0006] The multiple tube fermentation method is described in Recles etal., “Most Probable Number Techniques” published in “Compendium ofMethods for the Microbiological Examination of Foods”, 3rd ed. 1992, atpages 105-199, and in Greenberg et al., “Standard Methods For theExamination of Water and Wastewater” 8th ed. 1992). In this method, avolume of sample is dispensed into several tubes representing thisdilution range. The tubes are then incubated at the appropriatetemperature so that the bacteria in each tube are allowed to grow. Afterincubation at a specific temperature for a specific time, the number ofpositive tubes is counted. The most probable number can be determinedfrom the formula described in Recles et al., supra.

[0007] Water testing is mostly done by membrane filtration, where acertain volume of water is passed through the membrane and the membraneis incubated in a medium for a certain period of time. After appropriateincubation, the colonies are counted.

SUMMARY OF THE INVENTION

[0008] The present invention provides a simple method for more accuratequantification of the number of microorganism in a sample, or forquantification of any other type of discrete particulate biologicalmaterial within a sample. Such biological materials include fungi orother living organisms, as well as aggregates of proteins, such asenzymes, or even co-factors, using reaction mixtures well known to thosein the art. The invention generally makes use of a novel article whichis designed to hold a liquid sample in which chemical and/ormicrobiological reactants are provided. For example, such chemicalreactants may be a specific growth medium for bacteria. The device usedis generally in the form of an incubation plate having a multitude ofwells able to hold separate aliquots of liquid. Generally, the device isdesigned to hold between 5 and 100 ml of liquid in total, and the wellsare designed to form separate incubation chambers for each aliquot ofsample. The wells can be of same size or of different size and shape toincrease counting range and/or simulate dilution effects. See, Naqui etal., U.S. Ser. No. 08/201,110, filed Feb. 23, 1994, entitled “Apparatusand Method for Quantification of Biological Material in a LiquidSample”, incorporated by reference herein.

[0009] Thus, in a first aspect the invention features a method fordetection of a biological material in a sample. The method includes thesteps of liquifying the sample (if necessary) and pouring the liquifiedsample and reagent into the incubation plate. The incubation plate has agenerally flat horizontal surface and the surface is divided into aplurality of at least 20 recessed wells. Each well is adapted to hold analiquot of liquid and is sized and shaped, and formed of a suitablematerial, to hold the aliquot within the well by surface tension. Anyexcess liquid from the liquified sample is poured from the surface ofthe plate due to the hydrophobicity of the material used to form theplate. The method then involves incubating that incubation plate untilthe presence or absence of the biological material is determined. In apreferred embodiments the wells are chamfered to allow liquid, that isabove the horizontal plane, to be poured off easily (see FIG. 3).

[0010] As noted above, the biological material that can be detected isany material that forms a discrete particle, such as a microorganism,which may be quantified by determining the presence or absence of such abiological material within each well of the incubation plate. The samplemay be any biological sample or environmental sample such as wastewater, food, a surface swab, or swabs from other surfaces, such as athroat, or other samples well known to those in the art. This sample maybe a liquid sample, or may be dissolved in a liquid to form theliquified sample. Thus, the term “liquifying” in the above paragraphrefers to providing the sample in a liquid that once combined with amicrobiological reagent can be rapidly aliquoted within the incubationplate. The liquidified sample may remain as a liquid or may besolidified in the wells.

[0011] The incubation plate may be formed of any desired material, butin particular it is desirable that a plastic be used which allowsseparate aliquots of the liquified sample to be held by surface tensionwithin each well without cross contamination of the wells. Preferably,the material is hydrophobic. The surface can be untreated or treatedchemically or physically to enhance retention of liquid witning thewells.

[0012] The shape of the incubation plate is not relevant, and inpreferred embodiments is a generally circular shape (such as that of aPetri dish). Indeed, the incubation plate can be used to take the placeof a Petri dish. Specifically, the method of this invention can be usedto replace those existing tests that are generally run on Petri dishesto score the number of bacterial colonies. Since discrete aliquots ofthe sample are provided in the plate, one of ordinary skill in the artneed only score the number of positive wells in the plate to define thequantity of biological material within the original sample, as with-theMPN test discussed above.

[0013] The generally flat horizontal surface is designed to allow theliquid to be aliquoted readily between the wells and then excess liquidto be poured from the plate. In a preferred embodiment, a lip or pouringspout is provided for the plate. Those in the art will recognize thatthe depth and shape of the wells, as well as the material used to makethe wells and the plate, are chosen such that surface tension can beused to hold the aliquots within each well dependent on the type of theliquid used in the liquified sample.

[0014] In other preferred embodiments, the surface defines at least 40,60, 80 or even 200 or more recessed wells; the plate is formed of anyformable plastic; a lid is also provided to prevent contamination ofliquid within the wells; and the plate is provided in a sterile form sothat no positive aliquots are noted unless at least one biologicalmaterial particle is present in the sample.

[0015] In yet other preferred embodiments, the incubation plate is clearor colored, for example, white or yellow (to enhance the appearance ofcolor (e.g., blue)) within the incubation plate) and the well has adiameter of about 0.15 inches, and the plate a diameter of about 3 or 5inches.

[0016] In still other preferred embodiments, the incubation plate has a“pour-off pocket” adjacent to the surface of the plate. The pocket hassufficient capacity to contain the excess liquid to be removed from theplate surface. As an aid in preventing the excess fluid from spillingback onto the plate surface, it is preferable that the pocket contain anabsorbent material, e.g., a gauze-like material. In a particularembodiment, the plate has both a pour-off pocket and a “landing pad”.The “landing pad” is described below.

[0017] In a related aspect, the invention features a sterile incubationplate having a generally flat horizontal surface. The-surface defines aplurality of at least 20 recessed wells (in preferred embodiments, atleast 40, 60, 90 or even 200 recessed wells are provided) and each wellis adapted as described above to hold aliquots of liquid by surfacetension.

[0018] In preferred embodiments, the invention features the sterileincubation plate much as described above but having incorporated thereina “landing pad”, which is a generally central area of the plate lackingwells, which can receive the sample prior to that sample being dilutedin, for example, an incubation medium. Thus, a volume of 0.01 to 5 ml ofsample liquid may be applied in the “landing pad” area (depending on itssize and shape) and then that liquid dispensed into each well byapplying the diluent and growth supporting medium (e.g., the Colilert™chemical mixtures noted above) and that liquid will simultaneouslydilute the sample and allow dispersion of the sample throughout thewells.

[0019] In addition, a pour spout can be provided within the incubationplate to allow pouring-off excess liquid within the plate. Such a pourspout can be matched with a suitable lid having a slit which allowsliquid in the incubation plate to be poured from the incubation plateonly when the slit is lined up with the pour spout, as described below.

[0020] As indicated for the method above, the incubation plate may alsohave a “pour-off pocket” adjacent to the surface of the plate. Thepocket has sufficient-capacity to contain the excess liquid to beremoved from the plate surface, and preferably the pocket contains anabsorbent material, e.g., a gauze-like material. In a particularembodiment, the plate has both a “pour-off pocket” and a “landing pad”.

[0021] Applicant provides an extremely useful method which allowsunskilled personnel to rapidly determine the quantity of biologicalmaterial within a sample. Since the sample is readily liquified bypeople without significant training in microbiology, and the materialsfor any specific tests can be provided by the manufacturer, such peoplecan readily perform the tests with accuracy. The incubation plate isgenerally provided in the sterile form so that no inappropriatedetection of biological material can occur.

[0022] While it is known to provide plastic containers which can holdliquid within a plurality of recesses, applicant believes that thisdevice provides a new automatic aliquoting method. This is animprovement over the existing products used to detect and quantify,microorganisms because the liquid migrates to the individual wellswithout individual dispensing.

[0023] The present device can replace the use of a Petri dish and can beused particularly in food analysis and in testing of clinical samples.The separation of the wells of the present device prevents crosstalk orcontamination between each aliquot. Because of this, many of the testscan be performed by observing fluorescence (which is not readilyperformed in an agar-containing Petri dish). The device is particularlyuseful when there is a large quantity of microorganisms present in asample, such as more than one organism per one ml or per ten ml.

[0024] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0025] The drawing will first briefly be described.

DRAWINGS

[0026]FIGS. 1 and 2 are diagrammatic representations of a formed plasticincubation plate.

[0027]FIG. 3 shows a cross section of a well, with or without a chamfer.

[0028]FIG. 4 is a diagrammatic representation of a formed plasticincubation plate having a pour spout and corresponding slit as well as a“landing pad”.

[0029]FIG. 5 is a diagrammatic representation of a formed plasticincubation plate having a “landing pad” and a “pour-off pocket”.

STRUCTURE

[0030] Referring to FIGS. 1 and 2 there is shown an incubation plate 10having a plurality of wells 12 each having a diameter of about 0.16inches. The incubation plate 10 has a diameter of about 5 inches. Theincubation plate is made of formed plastic. Wells 12 are spaced apartsufficiently to prevent crosstalk between the wells, These wells mayhave a chamfer (FIG. 3) if desired to prevent liquid remaining at theupper edge of the well. Those in the art will recognize that incubationplate 10 can be readily formed by standard procedure and manufactured inthe general shape of a Petri dish, with or without a lip or pouringspout, and with or without a lid 14. This lid is provided with a dimple16 to prevent contact of the lid with plate 10.

[0031] Referring to FIG. 4, there is shown in incubation plate 10 havinga plurality of wells much as described above. The incubation plate alsoincludes a “landing pad” 22 of size about one and one-half inch diameterwhich is simply an area able to hold a defined volume of liquid. Withinthe incubation plate is also provided a pour spout 24 which allowsexcess liquid to be removed from the incubation plate. Also provided isa corresponding lid 14 having a slit which can be matched with the pourspout to allow liquid to be removed from the incubation plate. When theslit is not aligned with the pour spout, evaporation of liquid withinthe plate is decreased by reducing air flow over the liquid in thewells. A dimple 16 may also be provided in the lid to prevent the lidsurface contacting the wells and thus preventing cross contaminationbetween the wells.

[0032] Referring to FIG. 5, there is shown an incubation plate 10 havinga plurality of wells much as described above, which also includes a“landing pad” 22 of size about one inch diameter. Within the incubationplate is also provided a “pour-off pocket” 26 adjacent to the surface ofthe plate which allows excess liquid to be removed from the incubationplate. As shown in the cross-sectional view, the “pour-off pocket” isformed by an elevated barrier 28 between the pocket and the platesurface. The barrier has a lower barrier section 30, which serves as achannel through which the excess fluid from the plate surface may bepoured into the pocket. Typically, the pocket will contain an absorbentmaterial which will retain the fluid within the pocket, preventing backspill onto the plate surface.

USE

[0033] In use, a reagent powder suitable for detection of a biologicalmaterial can be rehydrated with an appropriate amount of sterile liquidand then inoculated with a known volume of a test sample. For example,20 ml of sterile water can be inoculated with between 10 and 1,000microliter of sample. The inoculated reagent can then be added toincubation plate 10 and that liquid swirled within incubation plate 10to distribute the inoculated liquid reagent to each of wells 12.Incubation plate 10 is then held at an angle of approximately 90 degreesto allow excess inoculated liquid reagent to be removed from the plate.A lid may then be placed on the incubation plate and that plate held inan incubator for the appropriate length of time, for example 18-48hours. After that length of time, the presence or absence of a positiveresult can be scored in each well 12 of the plate. In addition, forplates having a “pour-off pocket”, due to the larger volume of fluidcontained in the pocket, a positive result in the pocket can serve as anearly indication of high bacterial counts.

EXAMPLE 1 Use of Incubation Plate For Bulk Testing

[0034] For total plate count a plate as described above is used for thedetection and quantification of the total bacterial concentration offood. It is based on a Multiple Enzyme Technology (Townsend and Chen,Method and Composition for Detecting Bacterial Contamination in FoodProducts, U.S. Ser. No. 08/484,593 hereby incorporated by referenceherein) which correlates enzyme activity to the presence of viablebacteria in food. It utilizes multiple enzyme substrates that produce ablue fluorescent color when metabolized by bacteria. When the liquidreagent is inoculated with a prepared food sample and dispensed into aplate, as described herein the total viable bacterial concentration ofthat food product can be determined after 24 hours of incubation. Theactual medium used herein is not critical to the invention, but isprovided only for illustrative purposes.

[0035] Storage and Disposal

[0036] Store bulk powder and unused Simplates at room temperature (4 to25° C.) away from the light. After use, the Simplate device will containviable bacteria which must be handled and discarded appropriately. Oncethe powder is rehydrated it is stable for up to 24 hours when stored at4 to 25° C.

[0037] Test Procedure

[0038] 1. Pour an appropriate amount of bulk powder to a container ofsterile deionized water. One vial contains enough powder for 10 tests.Each test has a final volume of 10 ml. For example: add 1 vial of powderto 100 ml of sterile water to make enough media for 10 tests.

[0039] 2. Place test sample on the center “landing pad” 22 of the plate10 shown in FIG. 4. At the completion of this procedure half of the testsample will be poured off and discarded, therefore, the size of theinoculum must take this into account. For example, if you wish tomeasure the bacterial concentration of 0.1 ml of test sample then youmust place 0.2 ml of test sample on the “landing pad”. Place no morethan 2 ml on the center “landing pad”.

[0040] 3. Remove the lid from the plate and dispense 10 ml of TPC mediain the plate making sure to direct the liquid over the test sample onthe center “landing pad”. If the test sample is greater than 0.1 ml addenough TPC to achieve a final volume of 10 ml in the plate. Note, if theliquid is not dispensed on the “landing pad” it may splatter.

[0041] 4. Place the lid back-on the plate. Note, to ensure that theliquid remains in the Simplate make sure that the slit on the lid is notlined up with the pour spout.

[0042] 5. Distribute the liquid into the wells by swirling the plate asyou would a standard pour plate.

[0043] 6. Line up the slit on the lid with the pour spout and carefullypour off the excess liquid that did not end up in the wells. Holding theplate at an angle of approximately 90° from the work bench ensuresproper pour off of excess liquid. Make sure that all liquid “crossbridges” between wells are removed by gently tapping the plate. Disposeof excess liquid appropriately.

[0044] 7. Slide the lid away from the pour spout to avoid drying theliquid in the wells during incubation and to avoid contamination fromoutside through the opening.

[0045] 8. Place the plate in an incubator for 24 hours. Plates can beinverted if desired. Incubation temperatures greater than 37° C. are notrecommended.

[0046] 9. Count the number of fluorescent wells after 24 hours byplacing a 6 watt 36 nm UV light within five inches of the plate. Do notread plate before 24 hours. Results are stable to 48 hours.

[0047] 10. Compare the number of fluorescent wells to an MPN chart todetermine the most probable number of bacterial present in the plate.

[0048] Test Procedure Using Plate Having “Pour-Off Pocket”

[0049] 1. Pour an appropriate amount of bulk powder to a container ofsterile deionized water. One vial contains enough powder for 10 tests.Each test has a final volume of 10 ml. For example: add 1 vial of powderto 10 ml of sterile water to make enough media for 10 tests.

[0050] 2. Place test sample on the center “landing pad” 22 of the plate10 shown in FIG. 5. At the completion of this procedure half of the testsample will be poured off into the pocket, therefore, the size of theinoculum must take this into account. For example, if you wish tomeasure the bacterial concentration of 0.1 ml of test sample then youmust place 0.2 ml of test sample on the “landing pad”. Place no morethan 2 ml on the center “landing pad”.

[0051] 3. Remove the lid from the plate and dispense 10 ml of media inthe plate making sure to direct the liquid over the test sample on thecenter “landing pad”. If the test sample is greater than 0.1 ml addenough media to achieve a final volume of 10 ml in the plate. Note, ifthe liquid is not dispensed on the “landing pad” it may splatter.

[0052] 4. Distribute the liquid into the wells by swirling the plate asyou would a standard pour plate taking care that the fluid does notenter the “pour-off pocket”.

[0053] 5. Carefully pour off the excess liquid that did not end up inthe wells through the “pour-off pocket” barrier channel. Holding theplate at an angle of approximately 90° from the work bench ensuresproper pour off of excess liquid. Make sure that all liquid “crossbridges” between wells are removed by gently tapping the plate.

[0054] 6. Place the plate in an incubator for 24 hours. Plates can beinverted if desired if the “pour-off” pocket contains an absorbentmaterial.

[0055] 7. Count the number of fluorescent wells after 24 hours byplacing a 6 watt 36 nm TV light within five inches of the plate. Do notread plate before 24 hours. Results are stable to 48 hours.

[0056] 8. Compare the number of fluorescent wells to an MPN chart todetermine the most probable number of bacterial present in the plate.

EXAMPLE 2 Use of Incubation Plate for Unit Dose Testing

[0057] The plate and media described in Example 1 are used for thistest.

[0058] Test Procedure

[0059] 1. Add 10 ml of sterile water to the tube of predispensed powder.If greater than 0.1 ml of food sample is to be inoculated into the test,reduce the volume of sterile water appropriately to achieve a finalvolume of 10 ml in the tube.

[0060] 2. Inoculate the liquid reagent with the food sample beingtested.

[0061] 3. Shake tube several times to completely mix powder andinoculated food sample. Avoid excessive mixing which tends to foam upliquid reagent. Too much foam can complicate the distribution of theliquid into the plate. The rest of the procedure is as in Example 1.

[0062] Other embodiments are within the following claims.

What is claimed is:
 1. A method for detection of the amount of abiological material in a sample, comprising the steps of: liquifyingsaid sample if necessary, and pouring the liquified sample into anincubation plate having a generally flat horizontal surface, saidsurface defining a plurality of at least twenty recessed wells, eachwell being adapted to hold an aliquot of liquid and being sized andshaped, and formed of a material suitable to hold said aliquot withineach said well by surface tension or by gelling of said liquid; pouringoff any excess liquid from said incubation-plate, and incubating saidincubation plate until the presence or absence of said biologicalmaterial in one or more of said wells is determined so that the amountof said biological material can be determined.
 2. The method of claim 1,wherein said surface defines at least 40 recessed wells.
 3. The methodof claim 1, wherein said surface defines at least 60 recessed wells. 4.The method of claim 1, wherein said surface defines at least 90 recessedwells.
 5. The method of claim 1, wherein said surface defines at least200 recessed wells.
 6. The method of claim 1, wherein said plate isformed of plastic.
 7. The method of claim 6, wherein said plastic isPVC.
 8. The method of claim 6, wherein said plastic is formed of ahydrophobic material.
 9. The method of claim 1, wherein said plate isgenerally circular in shape.
 10. The method of claim 1, wherein saidplate is provided with a lid to prevent contamination of liquid in thewells.
 11. The method of claim 1, wherein said plate is clear.
 12. Themethod of claim 1, wherein said plate is colored.
 13. The method ofclaim 12, wherein the color is yellow.
 14. The method of claim 1,wherein each said well is about 0.15 inch in diameter.
 15. The method ofclaim 1, wherein each said well is chamfered to aid in the removal ofexcess liquid.
 16. The method of claim 1, wherein said plate is aboutthree inches in diameter.
 17. The method of claim 1, wherein said plateis about five inches diameter.
 18. The method of claim 1, wherein saidplate has a lip to aid removal of excess liquid not present in a wellfrom said plate.
 19. The method of claim 1, wherein said wells hold atotal of between five to 100 ml.
 20. The method of claim 1, wherein saidplate is sterile.
 21. The method of claim 1, wherein said plate furthercomprises a pour-off pocket adjacent to said surface, wherein saidpocket is adapted to contain said excess liquid, and wherein said excessliquid is poured from said surface of said incubation plate into saidpour-off pocket prior to incubation.
 22. The method of claim 21, whereinsaid pour-off pocket contains an absorbent material.
 23. The method ofclaim 1, wherein said plate comprises a “landing pad” area of sizesufficient to hold the liquified sample prior to dispersal of the sampleto said wells.
 24. The method of claim 23 wherein said method comprisesapplying said sample to said “landing pad” and subsequently providing afurther liquid to simultaneously dilute said sample and disperse saidsample into said wells.
 25. The method of claim 23, wherein said platefurther comprises a pour-off pocket adjacent to said surface, whereinsaid pocket is adapted to contain said excess liquid, and wherein saidexcess liquid is poured from said surface of said incubation plate intosaid pour-off pocket prior to incubation.
 26. The method of claim 1,wherein said incubation plate comprises a pour spout and wherein saidplate further comprises a lid having a slit adapted to cooperate withsaid pour spout to allow excess liquid to be poured from said incubationplate and to allow separation of said slit in said lid and said pourspout in said incubation plate to thereby permit incubation of theincubation plate without significant loss of liquid from said wells byevaporation.
 27. A sterile incubation plate for determination of theamount of a biological material having a generally flat horizontalsurface, said surface defining a plurality of at least twenty recessedwells, each well being adapted to hold an aliquot of liquid and beingsized and shaped, and formed of a material suitable to hold said aliquotwithin each said well by surface tension, wherein said incubation platewill not provide any positive response for said biological material inthe absence of a said biological material present in a sample applied tosaid plate.
 28. The plate of claim 27, wherein said surface defines atleast 40 recessed wells.
 29. The plate of claim 27, wherein said surfacedefines at least 60 recessed wells.
 30. The plate of claim 27, whereinsaid surface defines at least 90 recessed wells.
 31. The plate of claim27, wherein said surface defines at least 200 recessed wells.
 32. Theplate of claim 27, wherein said plate is formed of plastic.
 33. Theplate of claim 32, wherein said plastic is PVC.
 34. The plate of claim32, wherein said plastic is formed of a hydrophobic material.
 35. Theplate of claim 27, wherein said plate is generally circular in shape.36. The plate of claim 27, wherein said incubation plate is providedwith a lid to prevent contamination of liquid in said wells.
 37. Theplate of claim 27, wherein said plate is clear.
 38. The plate of claim27, wherein said plate is colored.
 39. The plate of claim 38, whereinthe color is white or yellow.
 40. The plate of claim 27, wherein eachsaid well is about 0.15 inch diameter.
 41. The plate of claim 27,wherein said plate is about three inches diameter.
 42. The plate ofclaim 27, wherein said plate is about five inches diameter.
 43. Themethod of claim 27, wherein said plate has a lid to allow removal ofexcess liquid not held in a well from the plate.
 44. The plate of claim27, wherein said wells hold a total of between five to 100 ml.
 45. Thesterile incubation plate of claim 27, further comprising a pour-offpocket adjacent to said surface of said plate, wherein said pocket isadapted to contain excess liquid removed from said surface of saidplate.
 46. The sterile incubation plate of claim 27, wherein said platecomprises a “landing pad” adapted to hold a sample prior to aliquotingof said sample to said wells.
 47. The sterile incubation plate of claim46, wherein said “landing pad” has a diameter of at least one inch. 48.The sterile incubation plate of claim 46, wherein said plate comprises apour spout.
 49. The sterile incubation plate of claim 48, wherein saidplate further comprises a lid-having a slit adapted to cooperate withsaid pour spout to allow removal of liquid said incubation plate. 50.The sterile incubation plate of claim 46, further comprising a pour-offpocket adjacent to said surface of said plate, wherein said pocket isadapted to contain excess liquid removed from said surface of saidplate.
 51. The sterile incubation plate of claim 50, further comprisingan absorbent material within said pour-off pocket.